The overall purpose of this project is to understand cellular mechanisms which coordinate gene expression during transitions between balanced growth and nutritional impoverishment. We remain particularly interested in the regulatory roles played by the structurally and metabolically related guanine nucleotides pppGpp and ppGpp that are analogs of GTP and GDP, respectively, but bear a 3'-pyrophosphate. These unusual nucleotides have been implicated as a regulatory signal in these adjustments. Past annual reports have described the relA and spoT genes involvement in the synthesis and degradation of (p)ppGpp. We also described manipulating (p)ppGpp levels at will. Last year's annual report described a first glimpse of the properties of cells lacking detectable ppGpp. This year, we have probed further into RNA polymerase rpoBC suppressors of one component of the pleiotropic phenotype associated with ppGpp-dependent functions. We have sequenced and devised a way to exploit the remaining gene (gpp) in the (p)ppGpp metabolic pathway so as to manipulate the relative abundance of pppGpp and ppGpp. This should allow us to dissect nucleotide-specific effects in the future. Prokaryotic genes sharing transcriptional regulation in an operon often show related functions. Accordingly, we have explored the downstream region of the spo operon at 83 min and sequenced, localized, and partially characterized recG, gltS, and probably gltC genes. We have also discovered that there are two genes involved in gpp function rather than one; gppA and gppB. The gppA gene was found to share an operon with a new gene, we call rhlB which shares sequence homology with the so-called DEAD Box family of RNA helicase genes. We have also isolated three more putative rhl genes with PCR techniques, sequenced portions of each, and mapped all five.